Cell-restricted transcriptional modulators play critical roles in the process of selective gene regulation during hematopoiesis. We have been investigating the molecular and biological function of Erythroid Kr|ppel-like Factor (EKLF). EKLF is a cell-restricted transcription factor that is essential for completion of the erythroid program. Transcriptional activation is accomplished by its interaction with and modification by coactivators that lead to changes in chromatin structure and the onset of transcription, a process best established at the adult ?-globin locus. We find that EKLF also interacts with components of the nucleosome and of the transcriptional initiation complex, and propose in Aim 1 to illuminate how these varied interactions are integrated and properly assembled, leading to epigenetic modifications as a result of EKLF's actions. However, EKLF's general role is becoming increasingly more complex, as it is also expressed in the megakaryocyte-erythroid progenitor and interacts with two types of corepressors to repress transcription under constrained cell environments. Acetylation and sumoylation of EKLF play critical roles in the protein-protein contacts that are important for these effects, and the experiments of Aim 2 will decipher how these interactions are manifested within native repression targets. Finally, significant levels of EKLF reside in the cytoplasm of the erythroid cell in a form that shows subtle biochemical and functional differences compared to its nuclear version. The experiments of Aim 3 will address whether EKLF plays a role outside of the nucleus by isolating and characterizing cytoplasmic EKLF complexes. These studies will be aided by the use of in vivo chromatin binding assays and EKLF rescue systems in primary or minimally manipulated cells. Elucidating EKLF's role in regulatory phenomena will continue to illuminate novel aspects of erythroid biology and the essential mechanisms by which a cell-restricted transcription factor can exert varied yet highly controlled influences on expression that lead to genetic and epigenetic changes. PUBLIC HEALTH RELEVANCE: This proposal focuses on a continuing investigation of EKLF structure/function and how its post- translational modifications, protein-protein interactions, and protein-DNA interactions relate to its ability to generate erythroid cell-specific control of gene expression.